Tuberculosis (TB) therapy requires at least 6 months of treatment with four drugs: isoniazid, rifampin, pyrazinamide, and ethambutol. However, with the evolution and spread of drug resistant Mycobacterium tuberculosis (Mtb), currently available therapies have become inadequate to control multi-drug resistant (MDR) TB. Therefore, the discovery of new antibiotics is critical for the long-term control of TB. Our lab has conducted two screens for inhibitors of the DosRST two-component signaling pathway using a hypoxia-inducible GFP reporter strain. To find DosRST-inhibitors, we screened for compounds that inhibited reporter GFP fluorescence (Class I inhibitors) but that did not inhibit growth, because DosRST is not required for growth under the screening conditions. The first screen was of the Harvard Medical School Institute of Chemistry and Cell Biology (ICCB) collection (~220,000 small molecules), which identified 3 verified DosRST inhibitors. We repeated the screen using the 340,000 compound NIH Molecular Libraries Program (MLP) collection, and identified 3 new inhibitors. Notably, both screens also identified numerous compounds that inhibit Mtb growth, but independent of DosRST, which we refer to as Class II inhibitors. These compounds are likely targeting pathways essential for Mtb growth and may include leads for new TB therapeutics. We have established a series of secondary assays, chemoinformatic pipelines and mutant screening platforms to confirm, classify, prioritize and define the mechanism of action of the Class II inhibitors. This pipeline was used to characterize Class II inhibitors from ICCB collection, however, we have not yet characterized the 1250 Class II hits from the MLP collection. The goal of this R03 is to confirm, classify and prioritize Mtb growth inhibitors from the MLP collection and initiate characterization studies. This work will define and prioritize new chemical scaffolds that inhibit known and new Mtb drug targets. The NIH provided us with 1250 cherry-pick compounds, arrayed in 384-well plates, suitable for 10 high throughput secondary assays. Using established, high-throughput assays, we will confirm the hits for phenotypic properties (Aim 1A), including: growth inhibition in vitro to confirm their activity; half-maximal effective concentration; eukaryotic cytotoxicity; and inhibition of Mtb growth in macrophages. The cherry-picks will also be tested for mechanistic properties (Aim 1B), including for resistance using a panel mutants in known resistance genes including mmpL3, katG, folC, mmpR5; and modulation of redox and pH homeostasis. With the support of a medicinal chemist and a defined chemoinformatics pipeline, the compounds will be prioritized based on biological and chemical properties (Aim 1C). Fresh powders from 100 prioritized hits will be purchased and confirmed for the tested activities and for synergistic interactions with TB drugs (Aim 1D). OVERALL IMPACT: This R03 project will deliver a new collection of 100 prioritized compounds that are suitable for continued preclinical studies as new TB therapeutics.